PEB3, a cell envelope protein, was identified as a target for use in an immunomagnetic isolation system (IMS). <i>Peb3</i> was cloned into <i>Escherichia coli</i> and expressed as a His-tagged construct (His.PEB3). Rabbits were then challenged with the purified construct to produce polyclonal antisera. Isolation of <i>C. jejuni </i>from a mixed culture (<i>E. coli</i> or <i>Arcobacter</i> spp. and <i>C. jejuni)</i> with polyclonal antisera was attempted but failed to capture whole <i>C. jejuni </i>cells. <i>C. jejuni</i> expresses two cell envelope associated polysaccharides: a short chain lipooligosaccharide (LOS) and a long chain lipid-linked polysaccharide, capsular polysaccharide (CPS). The Penner serotyping system has been shown to use CPS as its major discriminatory determinant. A novel molecular based typing system was devised based upon restriction fragments length polymorphism (RFLP) using the CPS locus as a discriminatory determinant. Primers for use in long template PCR (LT-PCR) were designed against the genes, <i>kpsD, kpsS, gmhA2</i> and <i>cj1430</i>. The theoretical maximum amplicon length obtainable in LT-PCR is ~40 kb; however, this is dependent on a number of factors including the %GC of the target amplicon. The low %GC of <i>C. jejuni</i> DNA prevented the amplification of the entire locus length (~41 kb) in one reaction and by experimentation the maximum amplifiable length was determined to be 25 kb. This maximum length was only achieved sporadically and the maximum length that could be reliably amplified was ~15 kb. Because of this limitation only the amplicon <i>kpsS-gmhA2 </i>was used in the typing scheme. Amplification was only achieved with the type strain, <i>C. jejuni</i> NCTC 11168. This is believed to be due to difficulties in amplifying low %GC targets with the Expand<sup>TM</sup> 20 kb plus long template system (Roche).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659906 |
| Date | January 2006 |
| Creators | Needham, Simon Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12711 |
Page generated in 0.0019 seconds